Methods and systems for point of use removal of sacrificial material

ABSTRACT

A method of manufacturing a sensor, the method including forming an array of chemically-sensitive field effect transistors (chemFETs), depositing a dielectric layer over the chemFETs in the array, depositing a protective layer over the dielectric layer, etching the dielectric layer and the protective layer to form cavities corresponding to sensing surfaces of the chemFETs, and removing the protective layer. The method further includes, etching the dielectric layer and the protective layer together to form cavities corresponding to sensing surfaces of the chemFETs. The protective layer is at least one of a polymer, photoresist material, noble metal, copper oxide, and zinc oxide. The protective layer is removed using at least one of sodium hydroxide, organic solvent, aqua regia, ammonium carbonate, hydrochloric acid, acetic acid, and phosphoric acid.

FIELD

This application generally relates to methods and systems for nucleicacid sequencing. More specifically, the specification relates to methodsand systems for processing and/or analyzing nucleic acid sequencing dataand/or signals.

BACKGROUND

Deoxyribonucleic acid, or DNA, is the genetic material in the nuclei ofall cells. DNA is made of chemical building blocks called nucleotides.Nucleotides comprise three parts: a phosphate group, a sugar group andone of four types of nitrogen bases. The four types of nitrogen basesfound in nucleotides are: adenine (A), thymine (T), cytosine (C) andguanine (G). To form a strand of DNA, nucleotides are linked intochains, with the phosphate and sugar groups alternating.

The structure of a DNA molecule was first described in 1953 by FrancisCrick and James D. Watson as two strands wound around each other in adouble helix to resemble a twisted ladder. The two strands of DNAcontain complementary information: A forms hydrogen bonds only with T, Conly with G. The precise order in which the four types of nitrogen bases(A, T, C, G) appear in the strand determines genetic characteristics ofall life forms. The process of determining the precise order ofnucleotides within a DNA molecule is termed DNA sequencing. DNAsequencing may be used to determine the sequence of individual genes,larger genetic regions (i.e. clusters of genes or operons), fullchromosomes or entire genomes. Depending on the methods used, sequencingmay provide the order of nucleotides in DNA or RNA isolated from cellsof animals, plants, bacteria, archaea, or virtually any other source ofgenetic information. The resulting sequences may be used by researchersin molecular biology or genetics to further scientific progress or maybe used by medical personnel to make treatment decisions.

Conventional methods of DNA sequencing comprise chemical (i.e.electrophoresis), optical, and electronic methods. As compared to othermethods for detecting a DNA sequence, electronic sequencing methodsdiffer from other sequencing technologies in that modified/labelednucleotides and/or optics/optical measurements are not necessary.Instead, electronic sequencing methods rely on ion or other reactionbyproducts to identify the relevant DNA sequence.

A type of electronic DNA sequencing, ion semiconductor sequencing is amethod of DNA sequencing based on the detection of ions (for example,hydrogen ions) that are released during the polymerization of DNA. Ionsemiconductor sequencing is a method of “sequencing by synthesis,”during which a complementary strand is built by incorporation (pairingof bases) based on the sequence of a template strand.

The incorporation of a deoxyribonucleotide triphosphate (dNTP) into agrowing DNA strand involves the formation of a covalent bond and therelease of pyrophosphate and a positively charged hydrogen ion. A dNTPwill only be incorporated if it is complementary to the leading unpairedtemplate nucleotide. Ion semiconductor sequencing leverages this processby detecting whether a hydrogen ion is released when a single species ofdNTP is provided to the reaction.

Hydrogen ions may be detected by providing an array of microwells on asemiconductor chip. Beneath the layer of microwells is an ion sensitivelayer, below which is an ion sensitive (ISFET) or a chemical sensitive(chemFET) sensor. Each microwell on the chip may contain a template DNAmolecule to be sequenced. Each microwell containing a template strandDNA molecule also contains a DNA polymerase. A DNA polymerase is acellular or viral enzyme that synthesizes DNA molecules from theirnucleotide building blocks. A microfluidics device may be used tointroduce a solution of unmodified A, T, C, or G dNTP into themicrowells one after the other and one at a time. If an introduced dNTPis complementary to the next unpaired nucleotide on the template strand,a biochemical reaction occurs (which includes the release of a hydrogenion) and the introduced dNTP is incorporated into the growingcomplementary strand by the DNA polymerase. If the introduced dNTP isnot complementary there is no incorporation and no biochemical reaction.The release of a hydrogen ion during incorporation causes a change inthe pH of the solution in the microwell. That change in the pH of thesolution can be detected/measured by the ISFET or chemFET sensor andtranslated into an electrical pulse.

Before the next cycle of dNTP is introduced into the microwells, themicrowells are flushed with a wash solution. Unattached dNTP moleculesare washed out during the flush cycle. The detected series of electricalpulses are transmitted from the chip to a computer and are translatedinto a DNA sequence. Because nucleotide incorporation events aremeasured directly by electronics, intermediate signal conversion is notrequired. Signal processing and DNA assembly can then be carried out insoftware.

The chip may be fabricated by taking advantage of conventionalsemiconductor technology. However, the release of a hydrogen ionproduces a small and transient signal that is difficult to measure. Tofurther complicate detection and nucleic acid sequencing, ion detectionis sensitive to various forms of contaminants on the chip surfaces.Accordingly, problems arise during manufacturing, packaging and exposureof the chip surfaces to the environment. Thus, there is a need forimproved methods and an apparatus to prevent contamination of the chipsurfaces for reliable ion detection and sequencing.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a partof the specification, illustrate one or more exemplary embodiments andserve to explain the principles of various exemplary embodiments. Thedrawings are exemplary and explanatory only and are not to be construedas limiting or restrictive in any way.

FIG. 1 illustrates components of a system for nucleic acid sequencingaccording to an exemplary embodiment.

FIG. 2 illustrates cross-sectional and expanded views of a flow cell fornucleic acid sequencing according to an exemplary embodiment.

FIGS. 3A-F are schematic representations of a method of manufacturing asensor with a protective layer according to an exemplary embodiment.

FIGS. 4A-C are schematic representations of a sensor with a protectivelayer according to an exemplary embodiment.

FIG. 5 is flow diagram for performing a method of manufacturing a sensorwith a protective layer according to an exemplary embodiment.

FIGS. 6A-B are schematic representations of a sensor with ion-sensinglayers according to different exemplary embodiments.

SUMMARY

The disclosure relates to methods of manufacturing a sensor for nucleicacid sequencing having a protective layer that prevents surfacecontamination of the sensor during various stages of fabrication. Thedisclosure further relates to a sensor for nucleic acid sequencingcomprising an array of chemically-sensitive field effect transistors(chemFETs) and a conformal protective layer over the sensing surfaces,sidewalls of the cavities, and top surface of the array of chemFETs. Theprotective layer over the array of chemFETs may be removed by an enduser before Nucleic acid sequencing is performed.

In one embodiment, the disclosure relates to a method of manufacturing asensor for Nucleic acid sequencing. The method comprises forming anarray of chemically-sensitive field effect transistors (chemFETs);depositing a dielectric layer over the chemFETs in the array; depositinga protective layer over the dielectric layer; etching the dielectriclayer and the protective layer to form cavities corresponding to sensingsurfaces of the chemFETs; and removing the protective layer. In anotherembodiment, the step of etching includes etching the dielectric layerand the protective layer together to form cavities corresponding tosensing surfaces of the chemFETs. In one embodiment, the protectivelayer is at least one of a polymer, photoresist material, noble metal,copper oxide, and zinc oxide. In another embodiment, the protectivelayer is removed using at least one of an acid, a base, and an oxidizingsolution. For example, the protective layer may be removed using atleast one of sodium hydroxide, organic solvent, aqua regia, ammoniumcarbonate, hydrochloric acid, acetic acid, and phosphoric acid. Inanother embodiment, the dielectric layer includes at least one ofsilicon oxide, silicon nitride and silicon oxynitride. In oneembodiment, the method further comprises patterning a photosensitiveetch mask and wherein the etching of the dielectric layer and theprotective layer is a photolithographic process.

In another embodiment, the disclosure relates to a sensor for Nucleicacid sequencing. The sensor comprises an array of chemically-sensitivefield effect transistors (chemFETs), each chemFET in the array having afloating gate structure including an upper surface; a dielectric layerover the upper surfaces of the floating gate structures of the chemFETsin the array, the dielectric layer including cavities extending to theupper surfaces of the floating gate structures and corresponding tosensing surfaces of the chemFETs; a conformal protective layer over thesensing surfaces, sidewalls of the cavities, and top surface of thearray. In one embodiment, the protective layer defines a removablelayer. In another embodiment, the protective layer is at least one of apolymer, photoresist, noble metal, copper oxide, and zinc oxide. Inanother embodiment, the protective layer is removed using at least oneof sodium hydroxide, organic solvent, aqua regia, ammonium carbonate,hydrochloric acid, acetic acid, and phosphoric. In another embodiment,the dielectric layer includes at least one of silicon oxide, siliconnitride and silicon oxynitride. In another embodiment, the sensingmaterial includes at least one of tantalum oxide, titanium oxide, andtitanium nitride. In another embodiment, the sensing material is a metaloxide or metal nitride selected from one or more of the group of Al2O3,Ta2O5, HfO3, WO3, ZrO2, TiO2 or mixtures thereof.

DETAILED DESCRIPTION

FIG. 1 illustrates components of a system for nucleic acid sequencingaccording to an exemplary embodiment. The components include a flow celland sensor array 100, a reference electrode 108, a plurality of reagents114, valve block 116, wash solution 110, valve 112, fluidics controller118, lines 120/122/126, passages 104/109/111, waste container 106, arraycontroller 124, and user interface 128. The flow cell and sensor array100 includes inlet 102, outlet 103, microwell array 107, and flowchamber 105 defining a flow path of reagents over the microwell array107. The reference electrode 108 may be of any suitable type or shape,including a concentric cylinder with a fluid passage or a wire insertedinto a lumen of passage 111. The reagents 114 may be driven through thefluid pathways, valves, and flow cell by pumps, gas pressure, or othersuitable methods, and may be discarded into the waste container 106after exiting the flow cell and sensor array 100.

Fluidics controller 118 may control driving forces for reagents 114 andthe operation of valve 112 and valve block 116 with suitable software.Microwell array 107 may include an array of defined spaces or reactionconfinement regions, such as microwells, for example, that isoperationally associated with a sensor array 100 so that, for example,each microwell has a sensor suitable for detecting an analyte orreaction property of interest. Microwell array 107 may preferably beintegrated with the sensor array as a single device or chip. The flowcell may have a variety of designs for controlling the path and flowrate of reagents over microwell array 107, and may be a microfluidicsdevice. Array controller 124 may provide bias voltages and timing andcontrol signals to the sensor, and collect and/or process outputsignals. User interface 128 may display information from the flow celland sensor array 100 as well as instrument settings and controls, andallow a user to enter or set instrument settings and controls.

In an exemplary embodiment, such a system may deliver reagents to theflow cell and sensor array in a predetermined sequence, forpredetermined durations, at predetermined flow rates, and may measurephysical and/or chemical parameters providing information about thestatus of one or more reactions taking place in defined spaces orreaction confinement regions, such as, for example, microwells (or inthe case of empty microwells, information about the physical and/orchemical environment therein). In an exemplary embodiment, the systemmay also control a temperature of the flow cell and sensor array so thatreactions take place and measurements are made at a known, andpreferably, a predetermined temperature.

In an exemplary embodiment, such a system may be configured to let asingle fluid or reagent contact reference electrode 108 throughout anentire multi-step reaction. Valve 112 may be shut to prevent any washsolution 110 from flowing into passage 109 as the reagents are flowing.Although the flow of wash solution may be stopped, there may still beuninterrupted fluid and electrical communication between the referenceelectrode 108, passage 109, and the sensor array 107. The distancebetween reference electrode 108 and the junction between passages 109and 111 may be selected so that little or no amount of the reagentsflowing in passage 109 and possibly diffusing into passage 111 reach thereference electrode 108. In an exemplary embodiment, wash solution 110may be selected as being in continuous contact with the referenceelectrode 108, which may be especially useful for multi-step reactionsusing frequent wash steps.

FIG. 2 is an illustration of expanded and cross-sectional views of anexemplary flow cell 200 and shows a portion of an exemplary flow chamber206. A reagent flow 208 flows across a surface of a microwell array 202,in which reagent flow 208 flows over the open ends of the microwells.Microwell array 202 and sensor array 205 together can form an integratedunit forming a bottom wall (or floor) of flow cell 200. Referenceelectrode 204 can be fluidly coupled to flow chamber 206. Further, flowcell cover 230 encapsulates flow chamber 206 to contain reagent flow 208within a confined region.

Example flow cell structures and associated components can be found inU.S. Pat. No. 7,948,015 (filed Dec. 14, 2007).

FIG. 2 also illustrates an expanded view of exemplary microwell 201,dielectric layer 210, and exemplary sensor 214. The volume, shape,aspect ratio (such as base width-to-well depth ratio), and otherdimensional characteristics of the microwells are design parameters thatdepend on a particular application, including the nature of the reactiontaking place, as well as the reagents, byproducts, and labelingtechniques (if any) that are employed. Sensor 214 can be anion-sensitive field-effect transistor (ISFET) with a floating gatestructure 218 having sensor plate 220 separated from the microwellinterior by ion-sensing layer 216. Ion-sensing layer 216 may cover theentire microwell or a portion thereof. Ion-sensing layer 216 may coversurfaces between microwells. (See, for example, FIGS. 6A-B). Ion-sensinglayer 216 may be a metal oxide layer such as, for example and withoutlimitation, silicon nitride, tantalum oxide, aluminum oxide, or acombination thereof.

Ion-sensing layer 216, particularly in a region above floating gatestructure 218 and sensor plate 220, can alter the electricalcharacteristics of the ISFET so as to modulate a current flowing througha conduction channel of the ISFET. That is, sensor 214 can be responsiveto (and generate an output signal related to) the amount of charge 224present on ion-sensing layer 216 opposite of sensor plate 220. Changesin charge 224 can cause changes in a current between source 221 anddrain 222 of the ISFET. In turn, the ISFET can be used to provide acurrent-based output signal or indirectly with additional circuitry toprovide a voltage-based output signal. Reactants, wash solutions, andother reagents can move in and out of the microwells by a diffusionmechanism 240.

In an embodiment, reactions carried out in microwell 201 can beanalytical reactions to identify or determine characteristics orproperties of an analyte of interest. Such reactions can generatedirectly or indirectly byproducts that affect the amount of chargeadjacent to sensor plate 220. If such byproducts are produced in smallamounts or rapidly decay or react with other constituents, then multiplecopies of the same analyte can be analyzed in microwell 201 at the sametime in order to increase the output signal ultimately generated. Forinstance, multiple copies of an analyte may be attached to solid phasesupport 212, either before or after deposition into a microwell. Thesolid phase support 212 may be a microparticle, nanoparticle, bead, orthe like. For nucleic acid analyte, multiple, connected copies may bemade by rolling circle amplification (RCA), exponential RCA, and othersimilar techniques, to produce an amplicon without the need of a solidsupport.

FIGS. 3A-F are schematic representations of a method of manufacturing asensor with a protective layer according to one embodiment. Sensor 314may be an ion sensitive (ISFET) or a chemical sensitive (chemFET) sensorwith a floating gate 318 having sensor plate 320 separated from themicrowell interior by an ion-sensing layer (not shown), and may bepredominantly responsive to (and generate an output signal related to)an amount of charge present on the ion-sensing layer opposite of sensorplate 320. Changes in the amount of charge cause changes in the currentbetween source 321 and drain 322 of sensor 314, which may be useddirectly to provide a current-based output signal or indirectly withadditional circuitry to provide a voltage output signal.

FIG. 3A shows a sensor 314 which may be an ion sensitive (ISFET) or achemical sensitive (chemFET). Substrate 300 may comprise a siliconwafer, or other relevant materials for CMOS fabrication as appropriate.

FIG. 3B shows dielectric layer 330 disposed on sensor 314. Dielectriclayer 330 may comprise silicon oxide, silicon nitride and siliconoxynitride.

FIG. 3C shows protective layer 340 disposed on dielectric layer 330.Protective layer 340 may comprise a polymer, photoresist material, noblemetal, copper oxide, or zinc oxide.

FIG. 3D shows sensor 314 after patterning of protective layer 340.

FIG. 3E shows cavity 350 extending to the upper surfaces of the floatinggate structures and corresponding to sensing surfaces of the chemFETsafter etching protective layer 340 and dielectric layer 330.

FIG. 3F shows sensor 314 with cavity 350 after removal of protectivelayer 340. Protective layer 340 may be removed using sodium hydroxide,organic solvent, aqua regia, ammonium carbonate, hydrochloric acid,acetic acid, and phosphoric acid, for example.

FIGS. 4A-C are schematic representations of a sensor with protectivelayer 440 according to one embodiment. Sensor 414 may be an ionsensitive (ISFET) or a chemical sensitive (chemFET) sensor 414 withfloating gate 418 having sensor plate 420 separated from the microwellinterior by an ion-sensing layer (not shown, see FIGS. 6A and 6B forexample), and may be predominantly responsive to (and generate an outputsignal related to) an amount of charge present on the ion-sensing layeropposite of sensor plate 420. Changes in the amount of charge causechanges in the current between source 421 and drain 422 of sensor 414,which may be used directly to provide a current-based output signal orindirectly with additional circuitry to provide a voltage output signal.

FIG. 4A shows sensor 414 with cavity 450 formed for use as a microwell,for example, as described above with reference to FIG. 1. Dielectriclayer 430 extends to the upper surface of the floating gate structure.Dielectric layer 430 may include at least one of silicon oxide, siliconnitride and silicon oxynitride. Substrate 400 may comprise a siliconwafer, or other relevant materials for CMOS fabrication as appropriate.

FIG. 4B shows a conformal protective layer 440 over the microwellstructure of sensor 414. Protective layer 440 may comprise a polymer,photoresist material, noble metal, copper oxide, or zinc oxide.

FIG. 4C shows sensor 414 with cavity 450 formed for use as a microwellafter removal of protective layer 440. Protective layer 440 serves toprotect sensor 414 from environmental contamination and is removedbefore performing nucleic acid sequencing according to an exemplaryembodiment. Protective layer 440 may be removed using at least one ofsodium hydroxide, organic solvent, aqua regia, ammonium carbonate,hydrochloric acid, acetic acid, or phosphoric acid.

FIG. 5 is flow diagram for performing a method of manufacturing a sensorwith a protective layer 500 according to one embodiment. Step 510includes forming an array of chemically-sensitive field effecttransistors (chemFETs). Step 520 includes depositing a dielectric layerover the chemFETs in the array. Step 530 includes depositing aprotective layer over the dielectric layer. Step 540 includes etchingthe dielectric layer and the protective layer to form cavitiescorresponding to sensing surfaces of the chemFETs. Step 550 includesremoving the protective layer. The protective layer serves to protectthe sensor from environmental contamination and is removed beforeperforming nucleic acid sequencing according to an exemplary embodiment.

In one embodiment, the dielectric layer and the protective layer areetched together to form cavities corresponding to sensing surfaces ofthe chemFETs. In one embodiment, the protective layer comprises apolymer, photoresist material, noble metal, copper oxide, or zinc oxide.In one embodiment, the protective layer is removed using at least one ofsodium hydroxide, organic solvent, aqua regia, ammonium carbonate,hydrochloric acid, acetic acid, or phosphoric acid. In one embodiment,the dielectric layer includes at least one of silicon oxide, siliconnitride and silicon oxynitride. In one embodiment, a further actincludes patterning a photosensitive etch mask, wherein the etching ofthe dielectric layer and the protective layer is a photolithographicprocess. In one embodiment, the protective layer removal includesremoval of photoresist residue contamination resulting from the etchingstep.

FIGS. 6A-B are schematic representations of exemplary sensors withcavity 650 formed for use as a microwell, for example, as describedabove with reference to FIG. 1. Sensor 614 may be an ion sensitive(ISFET) or a chemical sensitive (chemFET) sensor 614 with floating gate618 having sensor plate 620 separated from the microwell interior by anion-sensing layer 616, and may be predominantly responsive to (andgenerate an output signal related to) an amount of charge present on theion-sensing layer opposite of sensor plate 620. Changes in the amount ofcharge cause changes in the current between source 621 and drain 622 ofsensor 614, which may be used directly to provide a current-based outputsignal or indirectly with additional circuitry to provide a voltageoutput signal. Substrate 600 may comprise a silicon wafer, or otherrelevant materials for CMOS fabrication as appropriate.

The invention claimed is:
 1. A sensor, comprising: an array ofchemically-sensitive field effect transistors (chemFETs), each chemFETin the array having a floating gate structure including an uppersurface; a dielectric layer over the upper surfaces of the floating gatestructures of the chemFETs in the array, the dielectric layer includingcavities extending to the upper surfaces of the floating gate structuresand corresponding to sensing surfaces of the chemFETs; a conformalprotective layer over the sensing surfaces, sidewalls of the cavities,and top surface of the array.
 2. The sensor of claim 1, wherein theprotective layer defines a removable layer.
 3. The sensor of claim 1,wherein the protective layer is at least one of a polymer, photoresist,noble metal, copper oxide, and zinc oxide.
 4. The sensor of claim 1,wherein the protective layer is removed using at least one of sodiumhydroxide, organic solvent, aqua regia, ammonium carbonate, hydrochloricacid, acetic acid, and phosphoric.
 5. The sensor of claim 1, wherein thedielectric layer includes at least one of silicon oxide, silicon nitrideand silicon oxynitride.
 6. The sensor of claim 1, wherein the sensingsurface comprises at least one of tantalum oxide, titanium oxide, andtitanium nitride.
 7. The sensor of claim 1, wherein the sensing surfacecomprises a metal oxide or metal nitride selected from one or more ofthe group of Al₂O₃, Ta₂O₅, HfO₃, WO₃, ZrO₂, TiO₂ or mixtures thereof.